In the human body, various kinds of proteins or peptides which are involved in the growth and differentiation of the cells are expressed. Such proteins or peptides bind to receptors on the cell wall to induce the expression of various signaling molecules, thereby maintaining the homeostasis of the body. However, if the amount of such proteins or peptides is not maintained at suitable levels in the body, problems associated with homeostasis will arise due to the lack or overexpression of signaling molecules in the body, thus causing various problems. For example, if the level of a human growth hormone or an epidermal growth factor associated with wound healing is low, the body will not grow or the wound will not heal well.
R. Canfield (in the year 1962), F. Esch (in the year 1985), S. Cohen (in the year 1962) and D. Metcalf (in the year 1985) found lysozyme, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and granulocyte colony-stimulating factor (G-CSF), respectively, which are protein factors consisting of amino acids, and they also identified the amino acid sequence of each of the protein factors (R. Canfield, J. Biol. Chem. 238, 2698, 1963; F. Esch, Proc. Natl. Acad. Sci. USA 82, 6507, 1985; S. Cohen, J. Biol. Chem. 237, 1555, 1962; D. Metcalf, Science 229, 16, 1985).
Such protein factors are known to have effects on would healing (lysozyme/bFGF/EGF) and leukopoiesis (G-CSF), and thus can be used either as therapeutic agents against foot ulcer that is likely to occur in diabetic patients or as therapeutic agents against neutropenia following anticancer therapy.
However, such proteins are known to have a short half-life in blood and tissue. Thus, when these proteins are administered for therapeutic purposes, there is a serious problem in that their in vivo potency and stability are significantly low.
To solve such problems, in recent 10 years, an attempt to conjugate the biocompatible polymer polyethylene glycol (PEG) to proteins or peptides has been continuously made (G. Pasut & F. M. Veronese Prog. Polym. Sci. 32, 933, 2007; F. M. Veronese, Biomaterials 22, 405.2001; P. Bailon, Pharm. Sci. Tech. Today 1, 352, 1998).
Polyethylene glycol (PEG) is a highly biocompatible polymer that does not cause an immune response in vivo and is one of synthetic polymers approved by the US FDA. This synthetic polymer can be used to protein-polyethylene glycol conjugates having increased molecular weights. Thus, these conjugates can keep protein from being cleared by the process of filtration in the kidneys. Also, these conjugates exhibit the effect of inhibiting enzymatic protein degradation in vivo through the stealth effect of polyethylene glycol, thereby increasing the in vivo half-life and stability of the protein. Examples of proteins having increased potency, obtained using this method, include human growth hormone, erythropoietin, interferon, insulin, interleukin, calcitonin, etc.
However, when polyethylene glycol is used to increase the in vivo stability and half-life of a protein drug, there is a problem in that polyethylene glycol reacts with a plurality of binding sites of the protein drug, and thus a heterogeneous mixture of multi-PEGylated species is produced. In particular, the heterogeneous mixture of multi-PEGylated species causes many problems in determining the in vivo half-life and stability of drugs.
Meanwhile, even when a mono-PEGylated conjugate containing one polyethylene glycol molecule bound thereto is prepared under strictly controlled conditions, there is a problem in that the biological or in vivo activity of the drug is significantly influenced by the binding site thereof. To solve this problem, there has been an attempt to conjugate PEG to the N-terminal of proteins or peptides, since the active sites of most therapeutic proteins (EPO, G-CSF, growth hormone, etc.) are not located adjacent to the N-terminal. Specifically, this is because N-terminal PEGylation is a technique capable of minimizing the reduction in protein activation that is the biggest shortcoming of PEGylation.
Thus, to realize the N-terminal site-specific mono-PEGylation as described above, there have been various attempts to site-specifically conjugate mono-polyethylene glycol (mono-PEG) with the primary amine of the N-terminal amine group of proteins or peptides.
The polyethylene glycols used in these prior attempts were methoxypolyethylene glycol derivatives having attached to one end N-hydroxysuccinimide or N-succinimidyl propionate that reacts specifically with the primary amine (T. H. Kim, Biomaterials, 23, 2311, 2002; H. Lee, Pharm. Res., 19, 845, 2002). Mono-PEGylated conjugates prepared using succinimide polyethylene glycol derivatives are known to have a very small change in in vivo activity, but N-hydroxysuccinimide or N-succinimidyl propionate is rapidly hydrolyzed during conjugation with proteins or peptides to make it difficult to prepare protein or peptide conjugates, thus reducing the yield of the preparation of the protein or peptide conjugates.
In another attempt to use site-specific protein mutation (single mutation), the technology of performing PEGylation by introducing more than 95% of cysteine into the N-terminal of protein (peptide) drugs has been attempted. However, this technology has a problem in that, because the protein drugs are chemically modified, the bioactivity of the drugs is reduced due to the modification.
In addition, N-terminal specific PEGylation employing aldehyde-PEG was attempted. It is a method applied for the production Neulasta® (Amgen Inc.). However, the reaction conditions in this technology must be limited to acidic conditions (pH 4-6, usually pH 5.0-5.5), and the reducing agent NaBH4 must also be used together. Thus, the application of this technology is limited due to such limited reaction conditions.
Accordingly, the present inventors have found that, if a conjugate of a protein or peptide with a PEG derivative having a compound containing a catechol bound thereto is prepared, a process for preparing the conjugate will be simple, the resulting product will have strong resistance to hydrolysis, and a conjugate comprising a protein or peptide site-specifically mono-PEGylated at the N-terminal primary group can be produced in high yield, thereby completing the present invention.